Anti-alpaca IgG2b Fc tag single-domain antibody and application thereof

Abstract

The invention relates to a single-domain antibody of an anti-alpaca IgG2b Fc tag. The single-domain antibody has the following complementarity determining regions: CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is as shown in any one of SEQ ID NO. 31-60, the amino acid sequence of the CDR2 is as shown in any one of SEQ ID NO. 61-90, and the amino acid sequence of the CDR3 is as shown in any one of SEQ ID NO. 91-120. The anti-alpaca IgG2b Fc tag single-domain antibody is short in research and development period and high in antibody quality, has outstanding advantages in the aspects of antibody affinity and targeting specificity, small molecule size, stability and modifiability, production cost and the like, and has outstanding advantages in the aspect of antigen recognition specificity. In addition, the single-domain antibody also has very strong tissue penetrability, can more easily penetrate through tissues and cell barriers and go deep into diseased tissues to play a role, and also can tolerate temperature, pH value and chemical environment changes in a wider range, so that the single-domain antibody has advantages in various application scenes.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a single-domain antibody against alpaca IgG2b Fc tag and application thereof. Background Art

[0002] Alpacas have attracted much attention in the field of immunoglobulin research. The IgG antibodies in their bodies have unique properties and diverse functions. Traditionally, serum antibodies mainly include four subtypes: IgG1, IgG2, IgG3 and IgG4, while the main serum antibodies of alpacas include two types: IgG1 and IgG2. IgG1 is similar to the structure of traditional antibodies and contains 2 heavy chains and 2 light chains; IgG2 lacks the CH1 and light chain domains, so it is also called heavy chain antibody (hcAb). IgG1 plays a key role in the early stage of immune response. It can quickly recognize and bind to antigens, activate the complement system, promote the phagocytic clearance of pathogens by phagocytes, show high affinity for a variety of antigens, and make significant contributions in resisting primary infection. IgG2 is especially produced in large quantities during the secondary immune response, and can maintain a state of immune protection for a long time. IgG2a has outstanding advantages in activating the complement system, can efficiently trigger the complement cascade reaction to kill pathogens, has strong affinity for complex antigens, and has good precision binding ability. IgG2b focuses on immune regulation. It regulates the activity of immune cells by binding to Fc receptors on the surface of immune cells, such as stimulating B cell proliferation and differentiation and promoting antibody production. It is indispensable in maintaining the body's immune balance. It also has a high affinity for specific antigens and can work with other subtypes to enhance immune defense. These subtypes of alpaca IgG antibodies cooperate and complement each other to build a sophisticated and efficient immune defense system, which not only provides protection for the health of alpacas themselves, but also provides rich resources and inspiration for biomedical research. It shows potential application value in the fields of disease diagnosis, immunotherapy and vaccine development, and helps promote further development and innovation in related fields.

[0003] Alpaca IgG2b antibody contains an antigen binding fragment and an Fc fragment, of which the Fc fragment has been widely used in animal immunization experiments or in vivo studies as a tag for recombinant protein antigens. It can promote the dimerization of recombinant proteins to enhance immunogenicity (while the tag itself will not have strong immunogenicity), and promote the recombinant protein to be easy to express and purify, have good activity, long half-life, and good stability, which helps to obtain better antigen-specific immune responses. In addition, the protein fused with the alpaca IgG2b Fc tag is convenient for subsequent detection (binding to the secondary antibody through the Fc tag) and purification (using the Fc tag to bind to protein A or protein G affinity chromatography columns) and other operations. Therefore, the development of antibodies against alpaca IgG2b Fc has many important uses. In terms of detection and quantitative analysis, it can be used as a primary antibody or used to capture recombinant proteins containing the tag in experiments such as Western Blot and ELISA, determine the expression of the recombinant protein and quantify its concentration, and help optimize expression conditions and quality control. For the purification of recombinant proteins, it can be fixed to the chromatography column matrix, and the recombinant protein can be efficiently separated from the mixture by specific binding, thereby improving the purity of the protein to meet the needs of subsequent research. In the study of the immune response mechanism, it is helpful to analyze the intrinsic principle of the tag to enhance the immunogenicity of the recombinant protein, explore the interaction between the antibody, the tag and the recombinant protein, and provide a basis for the development of immunotherapy strategies. In the process of alpaca nanobody development, the binding status of the nanobody and the IgG2b Fc tag can be evaluated, the design and screening can be optimized, and the stability and distribution of the nanobody-recombinant protein complex in the body can be monitored, providing a key reference for the in vivo application of nanoantibodies and promoting the in-depth development of related research and applications.

[0004] Since Hamers et al. discovered heavy chain antibodies with naturally missing light chains in camel blood in 1993, single domain antibodies (sdAb) have gradually replaced other small antibodies and gradually become a hot spot in the research and development of new antibody drugs. Single domain antibodies, also known as nanobodies, are usually only about 15KDa, about one-tenth the size of traditional antibodies. They have disulfide bonds inside and a large number of hydrophilic residues on the surface, and have strong resistance to heat and pH. The lack of Fc segments and light chains of sdAb enables it to recognize hidden epitopes or small epitopes that traditional antibodies cannot recognize, and avoid complement reactions. In addition, single domain antibodies also have many advantages such as high stability, low toxicity, strong solubility, easy target screening, and easy direct expression in prokaryotic microorganisms, and good economy. Sequence homology analysis shows that the VHH germline gene sequence of camel sdAb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than humans, and CDR3 protrudes outward in the tertiary structure, so it is speculated that it has higher antigen binding specificity and affinity. Based on the excellent characteristics of alpaca IgG2b Fc tag, the development of single-domain antibodies against alpaca IgG2b Fc tag will better play its important functions and add icing on the cake.

[0005] With the continuous development of the biopharmaceutical industry, the demand for new and high-performance antibodies is increasing, but there are also many challenges that cannot be ignored in the specific production process, such as high cost and long preparation cycle. The number of alpacas raised is relatively limited, and the production and preparation process of their antibodies requires high technology and cost investment, resulting in a relatively high price for alpaca IgG2b Fc tag antibodies, which to a certain extent limits its popularity in some large-scale applications. Therefore, efficiently screening high-affinity anti-alpaca IgG2b Fc tag single domain antibodies to serve the field of biopharmaceutical diagnosis and treatment has broad and far-reaching significance. Summary of the invention

[0006] In view of this, a single-domain antibody against alpaca IgG2b Fc tag and its application are provided, which has a short research and development cycle and high antibody quality, and has outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, production cost, etc., and has strong tissue penetrability and stability, can more easily penetrate tissue and cell barriers, penetrate deep into the diseased tissue to exert its effect, and can also withstand a wide range of temperature, pH value and chemical environment changes.

[0007] A single domain antibody against alpaca IgG2b Fc tag, the single domain antibody having the following complementary determining regions: CDR1, CDR2 and CDR3; Among them, the amino acid sequence of CDR1 is shown in any one of SEQ ID NOs.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs.91-120.

[0008] Preferably, the complementarity determining region of the single domain antibody against alpaca IgG2b Fc tag is as shown in any one of the following (1)-(30): (1) CDR1 is shown in SEQ ID NO.31, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.91; (2) CDR1 is shown in SEQ ID NO.32, CDR2 is shown in SEQ ID NO.62, and CDR3 is shown in SEQ ID NO.92; (3) CDR1 is shown in SEQ ID NO.33, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.93; (4) CDR1 is shown in SEQ ID NO.34, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.94; (5) CDR1 is shown in SEQ ID NO.35, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO.95; (6) CDR1 is shown in SEQ ID NO.36, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.96; (7) CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.67, and CDR3 is shown in SEQ ID NO.97; (8) CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.98; (9) CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.69, and CDR3 is shown in SEQ ID NO.99; (10) CDR1 is shown in SEQ ID NO.40, CDR2 is shown in SEQ ID NO.70, and CDR3 is shown in SEQ ID NO.100; (11) CDR1 is shown in SEQ ID NO.41, CDR2 is shown in SEQ ID NO.71, and CDR3 is shown in SEQ ID NO.101; (12) CDR1 is shown in SEQ ID NO.42, CDR2 is shown in SEQ ID NO.72, and CDR3 is shown in SEQ ID NO.102; (13) CDR1 is shown in SEQ ID NO.43, CDR2 is shown in SEQ ID NO.73, and CDR3 is shown in SEQ ID NO.103; (14) CDR1 is shown in SEQ ID NO.44, CDR2 is shown in SEQ ID NO.74, and CDR3 is shown in SEQ ID NO.104; (15) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.75, and CDR3 is shown in SEQ ID NO.105; (16) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.76, and CDR3 is shown in SEQ ID NO.106; (17) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.77, and CDR3 is shown in SEQ ID NO.107; (18) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.78, and CDR3 is shown in SEQ ID NO.108; (19) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.79, and CDR3 is shown in SEQ ID NO.109; (20) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.80, and CDR3 is shown in SEQ ID NO.110; (21) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.81, and CDR3 is shown in SEQ ID NO.111; (22) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.82, and CDR3 is shown in SEQ ID NO.112; (23) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.83, and CDR3 is shown in SEQ ID NO.113; (24) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.84, and CDR3 is shown in SEQ ID NO.114; (25) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.85, and CDR3 is shown in SEQ ID NO.115; (26) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.86, and CDR3 is shown in SEQ ID NO.116; (27) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.117; (28) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.118; (29) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.119; (30) CDR1 is shown in SEQ ID NO.60, CDR2 is shown in SEQ ID NO.90, and CDR3 is shown in SEQ ID NO.120.

[0009] Preferably, the single-domain antibody against alpaca IgG2b Fc tag has the following framework regions: FR1, FR2, FR3, and FR4; wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NOs.121-150; the amino acid sequence of FR2 is shown in any one of SEQ ID NOs.151-180; the amino acid sequence of FR3 is shown in any one of SEQ ID NOs.181-210; and the amino acid sequence of FR4 is shown in any one of SEQ ID NOs.211-240.

[0010] Preferably, the framework region of the single domain antibody is as shown in any one of the following (31)-(60): (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.211; (32) FR1 is shown in SEQ ID NO.122, FR2 is shown in SEQ ID NO.152, FR3 is shown in SEQ ID NO.182; FR4 is shown in SEQ ID NO.212; (33) FR1 is shown in SEQ ID NO.123, FR2 is shown in SEQ ID NO.153, FR3 is shown in SEQ ID NO.183; FR4 is shown in SEQ ID NO.213; (34) FR1 is shown in SEQ ID NO.124, FR2 is shown in SEQ ID NO.154, FR3 is shown in SEQ ID NO.184; FR4 is shown in SEQ ID NO.214; (35) FR1 is shown in SEQ ID NO.125, FR2 is shown in SEQ ID NO.155, FR3 is shown in SEQ ID NO.185; FR4 is shown in SEQ ID NO.215; (36) FR1 is shown in SEQ ID NO.126, FR2 is shown in SEQ ID NO.156, FR3 is shown in SEQ ID NO.186; FR4 is shown in SEQ ID NO.216; (37) FR1 is shown in SEQ ID NO.127, FR2 is shown in SEQ ID NO.157, FR3 is shown in SEQ ID NO.187; FR4 is shown in SEQ ID NO.217; (38) FR1 is shown in SEQ ID NO.128, FR2 is shown in SEQ ID NO.158, FR3 is shown in SEQ ID NO.188; FR4 is shown in SEQ ID NO.218; (39) FR1 is shown in SEQ ID NO.129, FR2 is shown in SEQ ID NO.159, FR3 is shown in SEQ ID NO.189; FR4 is shown in SEQ ID NO.219; (40) FR1 is shown in SEQ ID NO.130, FR2 is shown in SEQ ID NO.160, FR3 is shown in SEQ ID NO.190; FR4 is shown in SEQ ID NO.220; (41) FR1 is shown in SEQ ID NO.131, FR2 is shown in SEQ ID NO.161, FR3 is shown in SEQ ID NO.191; FR4 is shown in SEQ ID NO.221; (42) FR1 is shown in SEQ ID NO.132, FR2 is shown in SEQ ID NO.162, FR3 is shown in SEQ ID NO.192; FR4 is shown in SEQ ID NO.222; (43) FR1 is shown in SEQ ID NO.133, FR2 is shown in SEQ ID NO.163, FR3 is shown in SEQ ID NO.193; FR4 is shown in SEQ ID NO.223; (44) FR1 is shown in SEQ ID NO.134, FR2 is shown in SEQ ID NO.164, FR3 is shown in SEQ ID NO.194; FR4 is shown in SEQ ID NO.224; (45) FR1 is shown in SEQ ID NO.135, FR2 is shown in SEQ ID NO.165, FR3 is shown in SEQ ID NO.195; FR4 is shown in SEQ ID NO.225; (46) FR1 is shown in SEQ ID NO.136, FR2 is shown in SEQ ID NO.166, FR3 is shown in SEQ ID NO.196; FR4 is shown in SEQ ID NO.226; (47) FR1 is shown in SEQ ID NO.137, FR2 is shown in SEQ ID NO.167, FR3 is shown in SEQ ID NO.197; FR4 is shown in SEQ ID NO.227; (48) FR1 is shown in SEQ ID NO.138, FR2 is shown in SEQ ID NO.168, FR3 is shown in SEQ ID NO.198; FR4 is shown in SEQ ID NO.228; (49) FR1 is shown in SEQ ID NO.139, FR2 is shown in SEQ ID NO.169, FR3 is shown in SEQ ID NO.199; FR4 is shown in SEQ ID NO.229; (50) FR1 is shown in SEQ ID NO.140, FR2 is shown in SEQ ID NO.170, FR3 is shown in SEQ ID NO.200; FR4 is shown in SEQ ID NO.230; (51) FR1 is shown in SEQ ID NO.141, FR2 is shown in SEQ ID NO.171, FR3 is shown in SEQ ID NO.201; FR4 is shown in SEQ ID NO.231; (52) FR1 is shown in SEQ ID NO.142, FR2 is shown in SEQ ID NO.172, FR3 is shown in SEQ ID NO.202; FR4 is shown in SEQ ID NO.232; (53) FR1 is shown in SEQ ID NO.143, FR2 is shown in SEQ ID NO.173, FR3 is shown in SEQ ID NO.203; FR4 is shown in SEQ ID NO.233; (54) FR1 is shown in SEQ ID NO.144, FR2 is shown in SEQ ID NO.174, FR3 is shown in SEQ ID NO.204; FR4 is shown in SEQ ID NO.234; (55) FR1 is shown in SEQ ID NO.145, FR2 is shown in SEQ ID NO.175, FR3 is shown in SEQ ID NO.205; FR4 is shown in SEQ ID NO.235; (56) FR1 is shown in SEQ ID NO.146, FR2 is shown in SEQ ID NO.176, FR3 is shown in SEQ ID NO.206; FR4 is shown in SEQ ID NO.236; (57) FR1 is shown in SEQ ID NO.147, FR2 is shown in SEQ ID NO.177, FR3 is shown in SEQ ID NO.207; FR4 is shown in SEQ ID NO.237; (58) FR1 is shown in SEQ ID NO.148, FR2 is shown in SEQ ID NO.178, FR3 is shown in SEQ ID NO.208; FR4 is shown in SEQ ID NO.238; (59) FR1 is shown in SEQ ID NO.149, FR2 is shown in SEQ ID NO.179, FR3 is shown in SEQ ID NO.209; FR4 is shown in SEQ ID NO.239; (60) FR1 is shown in SEQ ID NO.150, FR2 is shown in SEQ ID NO.180, FR3 is shown in SEQ ID NO.210; FR4 is shown in SEQ ID NO.240.

[0011] Preferably, the amino acid sequence of the single-domain antibody is as shown in any one of SEQ ID NOs. 1-30.

[0012] Preferably, the framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody.

[0013] Preferably, the single-domain antibody against alpaca IgG2b Fc tag is prepared by immunizing camels with alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells of the immunized camel, isolating alpaca IgG2b Fc affinity lymphocytes therefrom, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the alpaca IgG2b Fc antigen by phage display technology, and verifying the binding force of the obtained single-domain antibody by enzyme-linked immunosorbent assay, thereby screening out a single-domain antibody against alpaca IgG2b Fc tag with high affinity activity.

[0014] Preferably, the DNA sequence of the single-domain antibody against alpaca IgG2b Fc tag is as shown in any one of SEQ ID NOs. 241-270.

[0015] Another aspect of the present application provides a fusion protein comprising the single domain antibody against alpaca IgG2b Fc tag as described above.

[0016] Furthermore, the present application also provides a product, which contains the single-domain antibody with anti-alpaca IgG2b Fc tag as described above or the fusion protein as described above, and the product is a diagnostic reagent, an in vitro diagnostic reagent, an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to a site expressing alpaca IgG2b Fc.

[0017] This application uses alpaca IgG2b Fc recombinant protein antigen to immunize camels, and successfully screens out alpaca IgG2b Fc single domain antibodies with high affinity activity. The heavy chain variable region of the single domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, single domain antibodies have higher antigen binding specificity and affinity than traditional antibodies. In addition, the single domain antibody and its application against alpaca IgG2b Fc tag of the present application have a short research and development cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost. In addition, the alpaca IgG2b Fc single domain antibody also has strong tissue penetration and stability, can more easily penetrate tissue and cell barriers, penetrate deep into the diseased tissue to play a role, and can also tolerate a wide range of temperature, pH value and chemical environment changes. It can still maintain its antigen binding activity in complex environments inside and outside the body, which makes it have advantages in a variety of application scenarios (such as in vivo treatment, in vitro diagnostic reagent development, etc.). It has more advantages in the early diagnosis of diseases (especially in cases where rapid and highly sensitive detection is required), targeted imaging and treatment of tumors (due to its good tissue penetration), and some research and application scenarios that require precise identification of subtle differences in antigens, such as the development of highly specific diagnostic reagents and new targeted therapeutic drugs.

[0018] In addition, the present invention provides a preparation scheme for the above-mentioned alpaca IgG2b Fc single domain antibody supernatant, expresses the alpaca IgG2bFc single domain antibody in small quantities, effectively reducing the development and production costs of the alpaca IgG2b Fc antibody, and the single domain antibody is verified by the ELISA system to have high specificity and high affinity for targeting alpaca IgG2b Fc, indicating that the alpaca IgG2b Fc single domain antibody obtained by the present invention has further development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the titer test results of camel immune serum against alpaca IgG2b Fc tag single domain antibody.

[0020] Figure 2 This is a schematic diagram of the results of detecting PCR products using agarose gel electrophoresis.

[0021] Figure 3 This is a graph showing the results of panning the affinity alpaca IgG2b Fc single domain antibody phage library.

[0022] Figure 4 This is an ELISA test result diagram for evaluating the enrichment degree of specific antibodies.

[0023] Figure 5This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-1-44.

[0024] Figure 6 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-45-136.

[0025] Figure 7 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-137-228.

[0026] Figure 8 This is a screen result of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the result of ELISA-229-312.

[0027] Fig. 9 This is a screen result of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the result of ELISA-313-395.

[0028] Fig.10 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-396-475.

[0029] Fig.11 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-476-486.

[0030] Fig.12 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-487-528.

[0031] Fig.13 This is a graph showing the screening results of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-529-572.

[0032] Fig.14 This is a screen result of positive clones of single-domain antibodies specific to alpaca IgG2b Fc, showing the results of ELISA-573-582.

[0033] Fig.15 Unique sequence analysis results of positive clones are shown.

[0034] Fig.16 Phage supernatant-based ELISA validation results are shown.

[0035] Fig.17The ELISA validation results based on the periplasmic expression product (VHH nanobody) are shown. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0037] The embodiment of the present invention provides a single domain antibody against alpaca IgG2b Fc tag, characterized in that the single domain antibody has the following complementary determining regions: CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is shown in any one of SEQ ID NO.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NO.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NO.91-120. Preferably, the complementary determining regions of the single domain antibody against alpaca IgG2b Fc tag, CDR1, CDR2, CDR3, each group matches as shown in the sequence table. Preferably, the single-domain antibody of the anti-alpaca IgG2bFc tag has the following heavy chain framework regions, namely FR1, FR2, FR3, and FR4; wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NO.121-150; the amino acid sequence of FR2 is shown in any one of SEQ ID NO.151-180; the amino acid sequence of FR3 is shown in any one of SEQ ID NO.181-210; the amino acid sequence of FR4 is shown in any one of SEQ ID NO.211-240. Preferably, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO.1-30. Preferably, the framework region is a heavy chain framework region, and at least a part of the heavy chain framework region is independently derived from an alpaca antibody. Preferably, the DNA sequence of the single-domain antibody of the anti-alpaca IgG2b Fc tag is shown in any one of SEQ ID NO.241-270.

[0038] Compared with the traditional alpaca IgG2b Fc-tagged antibody, the functional characteristics of the alpaca IgG2b Fc-tagged single-domain antibody are mainly manifested in the following aspects. First, the advantages in antigen recognition specificity are outstanding. The VHH domain of the alpaca IgG2b Fc-tagged single-domain antibody has a highly specific antigen recognition ability. It can recognize and bind to specific epitopes on the antigen. This binding is based on the amino acid sequence complementarity and spatial structure complementarity of the VHH domain, forming a tight non-covalent binding with the antigen. Because the structure of the single-domain antibody is simpler, the epitope that binds to the antigen may be difficult for traditional antibodies to access, such as some epitopes hidden inside the antigen molecule, which provides the possibility for the discovery and application of new antigen targets. Second, it can bind to the antigen with high affinity. Although there is only one antigen binding site, the VHH domain of the single-domain antibody can bind to the antigen with a high affinity. Its affinity constant (Kd) can usually reach 10⁻ 7 - 10⁻ 9 M or even lower levels, can achieve effective antigen binding at lower antibody concentrations, which is of great significance in disease diagnosis and treatment, for example, it can be used to develop highly sensitive diagnostic reagents or highly effective therapeutic antibodies. Although the affinity of a single binding site may be relatively low, it has advantages in identifying certain hidden antigen epitopes that are difficult for traditional antibodies to access. Moreover, its binding specificity is strong, and it can more accurately identify subtle differences in antigens, such as distinguishing different isomers or mutants of antigens. Third, it has strong tissue penetration and stability. Due to its small molecular weight, the alpaca IgG2b Fc-tagged single domain antibody has good tissue penetration, and can more easily penetrate tissue and cell barriers and penetrate deep into the diseased tissue to play a role. Fourth, it also has high stability, can withstand a wide range of temperature, pH and chemical environment changes, and can still maintain its antigen binding activity in complex environments inside and outside the body, which makes it advantageous in a variety of application scenarios (such as in vivo treatment, in vitro diagnostic reagent development, etc.). It has more advantages in the early diagnosis of diseases (especially in cases where rapid and highly sensitive detection is required), targeted imaging and treatment of tumors (due to its good tissue penetration), and some research and application scenarios that require precise identification of subtle differences in antigens, such as the development of highly specific diagnostic reagents and new targeted therapeutic drugs.

[0039] Preferably, the single-domain antibody against alpaca IgG2b Fc tag is prepared by immunizing camels with alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells of the immunized camel, isolating alpaca IgG2b Fc affinity lymphocytes therefrom, extracting total RNA and reverse transcribing it into cDNA, cloning the V region of the camel heavy chain antibody using Nest-PCR technology, inserting it into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the alpaca IgG2b Fc antigen by phage display technology, and verifying the binding force of the obtained single-domain antibody by enzyme-linked immunosorbent assay, thereby screening out a single-domain antibody against alpaca IgG2b Fc tag with high affinity activity.

[0040] Therefore, the screening of single domain antibodies against alpaca IgG2b Fc tag of the present application is divided into the following stages: ① camel immunization and serum titer determination; ② camel single domain antibody phage display library construction; ③ camel single domain antibody phage display library amplification and rescue; ④ selection of single domain antibodies specifically binding to alpaca IgG2b Fc using phage display technology; ⑤ identification of single domain antibody positive clones specific to alpaca IgG2b Fc; ⑥ positive clone sequence analysis; ⑦ verification of unique clones that specifically bind to alpaca IgG2b Fc.

[0041] The above-mentioned stages are illustrated below by means of specific embodiments.

[0042] ① Camel immunization and serum titer determination (1) Alpaca IgG2b Fc immunization of camel 1 mg of alpaca IgG2b Fc was mixed with an equal volume of Freund's adjuvant and injected at 3-5 points under the skin of the camel's neck. Blood was collected from the ear vein of the camel before immunization. Immunization was performed once every two weeks, for a total of 5 times; 5 mL of peripheral blood was collected from the camel during each immunization. When collecting blood, the camel's head was fixed to one side, the skin of the animal's blood collection site was shaved first, and 75% alcohol was disinfected. After drying, blood was collected. The jugular vein groove was pressed with a finger. After the blood vessel was dilated, the needle was disinfected at the blood collection site to collect blood. 5 mL of blood was collected and left to stand for the preparation of serum for titer evaluation. 7 days after the last immunization, 50 mL of blood was collected in an EDTA anticoagulant tube, and it was immediately shaken continuously and slowly, mixed thoroughly, placed on ice, and transported back to the laboratory.

[0043] (2) Serum titer detection 100 ng of alpaca IgG2b Fc antigen was coated on a 96-well high-adsorption ELISA plate at 4 °C overnight. The coated antigen was washed 3 times with PBST, and then blocked with 200 uL 2% BSA at room temperature for 2 hours. The serum collected before and after each immunization was diluted to different concentration gradients, and the blocking solution was discarded. The ELISA plate was washed 3-5 times with PBST, and 100 uL of serum samples with different dilution gradients were added to each well, incubated at room temperature for 2 hours, and then the serum was discarded, and washed 5 times with PBST. CamelidIgG-HRP was diluted with 1% BSA, and 100 uL was added to each well. Incubated at room temperature for 1.5 hours in the dark, and then the plate was washed 5 times. 100 uL TMB colorimetric solution was added to each well, incubated at room temperature for 10-30 min, and OD450 readings were taken after adding the stop solution. The results of serum titer detection are attached. Figure 1 .

[0044] (3) Separation of blood lymphocyte samples Lymphocytes were isolated from blood samples collected after the last immunization using the following method: i. Add 7 mL of Ficoll, a lymphocyte separation medium, to each 15 mL centrifuge tube; ii. Add an equal volume of PBS (1×) or saline to the fresh whole blood to which the anticoagulant (EDTA) has been added and mix thoroughly; iii. Take the 15 mL centrifuge tube with lymphocyte separation solution and slowly and carefully transfer it to another 15 mL centrifuge tube with lymphocyte separation solution, and make the mixed solution above the surface of lymphocyte separation solution (i.e., the two liquids should not be mixed and a clear interface should be retained), and centrifuge at 3,000 g for 20 min; iv. Use a 1 mL pipette to carefully transfer the supernatant to a 1.5 mL cell cryopreservation tube, write the animal number and plasma, put it in a small cloth bag with a rope, and store it in a liquid nitrogen tank. Use a 1 mL pipette to carefully separate the white blood cell layer into a 15 mL centrifuge tube; fill it up to 15 mL with PBS (1×); wash the white blood cells with PBS (1×), centrifuge (3,000 g, centrifuge for 20 min), carefully pour off the supernatant, do not stir the cell clumps at the bottom of the tube, and recover the white blood cells in the remaining 0.1 - 0.2 mL PBS.

[0045] vi. Add 5 times the volume of RNA later, gently dissolve the cell clumps, divide into 2 portions into 1.5 mL cell cryopreservation tubes, and store in liquid nitrogen tank.

[0046] ② Construction of camel single domain antibody phage display library (1) Total RNA extraction Take a portion of frozen lymphocytes, add 1 mL Trizol, let it stand at room temperature for 10 min, then add 0.2 mL chloroform, shake vigorously, let it stand at room temperature, wait for the solution to separate (about 10 min), centrifuge at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix, let it stand at room temperature for 15 min, wait for the nucleic acid to precipitate, centrifuge at high speed to remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge at high speed to remove the supernatant, control the water, dissolve the RNA in nuclease-free water, and take 1 uL for concentration and purity determination.

[0047] (2) cDNA synthesis 1 ug RNA was taken and cDNA was synthesized using the cDNA first-strand synthesis kit (Super Script TMIII First-StrandSynthesis SuperMix (Invitrogen)). Oligo dT was used as the reverse transcription primer and the synthesized cDNA was frozen at -20°C; (3) Phage display library construction and PCR amplification The above synthesized cDNA was used as a template to amplify the V region (VHH) of the camel heavy chain antibody using Nest-PCR. The following table lists the names and sequences of the Nest-PCR primers:

[0048] a. First round PCR reaction system: cDNA 1 uL; Mix 12.5 uL; CALL001 0.5 uL; CALL002 0.5 uL; water to 25 uL. First round PCR reaction conditions: 95 ℃ 5 min; 94 ℃ 1 min, 57 ℃ 1 min, 72 ℃ 1 min, 35 cycles; 72 ℃ 5 min. b. Second round PCR reaction system: 40 ng of the first round PCR product; 25 uL of Mix; 1 uL of VHH-Back; 1 uL of VHH-For; water to 50 uL. Second round PCR reaction conditions: 95 ℃ 5min; 94 ℃ 45 s, 60 ℃ 45 s, 72 ℃ 45 s, 15 cycles; 72 ℃ 5 min. c. After the PCR reaction, the PCR product was detected by 1.5% agarose gel electrophoresis. The target gene fragment of the first round of PCR was at 700 bp. The gel was cut and the target band was recovered by the QIAEX II Gel Extraction kit. The second round of PCR was performed. The target gene fragment was at 500 bp. The target band was recovered by cutting the gel, i.e., the VHH fragment. The electrophoresis results are attached. Figure 2 .

[0049] (4) Phagemid vector digestion and target fragment ligation The VHH fragment and pMES4 vector were double-digested with restriction endonucleases Eco91I and PstI, respectively. The reaction system was as follows: a. Vector enzyme digestion system: pMES4 vector 20 ug; PstI 10 uL; Eco91I 20 uL; Cutsmartbuffer 50 uL; add H2O to 500 uL. b. Fragment digestion system: VHH fragment 5 ug; PstI 7 uL; Eco91I 14 uL; Cutsmart buffer 50 uL; add H2O to 500 uL. Digest overnight at 37 ℃, and after agarose gel electrophoresis, cut the gel for recovery; mix the digestion products of the vector and VHH fragment, and connect them with T4 DNA Ligase at 16 ℃ overnight.

[0050] ③ Amplification and rescue of camel single domain antibody phage display library (1) Construction of phage display library After the ligation product was purified by PCR Purification Kit, 1 uL was taken to transform E. coli TG1 competent cells, revived at 37 ℃ for 2 h, gradiently diluted to 101, 102, and 103, and 300 uL was taken to coat the plate, cultured at 37 ℃ overnight, and the number of clones was calculated, about 105 clones / plate. The same transformation method was used for large-scale transformation until the number of clones in the library reached more than 108. All clones were eluted with LB, centrifuged at 5,000g for 5 min, and the precipitate was suspended with 2 mL LB, an equal volume of 30% glycerol was added, and frozen at -80 ℃.

[0051] (2) Library diversity detection 80 clones of (1) were randomly selected for sequencing to detect the recombination rate and evaluate the quality of the library. The recombination rate of the constructed alpaca IgG2b Fc single domain antibody library was 92.5%. The diversity of the alpaca IgG2b Fc single domain antibody library was analyzed. The sequencing results showed that 64 monoclones had 54 kinds of amino acid sequences, indicating that the constructed library had good diversity.

[0052] (3) Phage amplification and rescue The phage library of alpaca IgG2b Fc single domain antibody was amplified and rescued using helper phage. The preserved monoclonal library was inoculated into 100 mL of culture medium and cultured to the logarithmic growth phase. Helper phage with an MOI of 20 was added. The precipitate was suspended with culture medium at room temperature for 30 min. After low-speed centrifugation, the precipitate was suspended with culture medium and inoculated into 300 mL of culture medium and cultured overnight. The next day, centrifuged at 3,000g for 30 min, the supernatant was collected, PEG was added to precipitate the phage, the precipitate was incubated on ice for 30 min, and 3,000g was centrifuged for 30 min. The precipitate was the alpaca IgG2b Fc single domain antibody phage library. After suspending the precipitate with PBS, the titer was determined to be 2 x1013 pfu / mL.

[0053] ④ Use phage display technology to select single-domain antibodies that specifically bind to alpaca IgG2b Fc (1) Affinity alpaca IgG2b Fc single domain antibody phage library panning A total of 4 rounds of panning were performed, and the alpaca IgG2b Fc antigen was used to coat the ELISA plate and incubated overnight at 4°C. The next day, the rescued alpaca IgG2b Fc single domain antibody phage was added and incubated at room temperature for 2 h; the wells were washed 10 times with PBST, 100 uL of triethylamine was added, and incubated at room temperature for 30 min. The collected phages were the alpaca IgG2b Fc single domain antibody phage library obtained by affinity panning; 10 uL of infected TG1 cells were taken to coat the plate for determination of the number of clones after screening, and the remaining screened phages were used for amplification. The results of each round of panning are attached. Figure 3 .

[0054] (2) Amplification and rescue of phage after screening The amplification and rescue methods are the same as above. The obtained PBS suspension, i.e. the phage after the first round of screening, is stored at 4°C and used for the next round of screening. According to the same screening steps as above, the amount of antigen is gradually reduced for 3-4 rounds of screening.

[0055] (3) ELISA to evaluate the enrichment of specific antibodies ELISA plates were coated with 100 ng of alpaca IgG2b Fc antigen at 4°C overnight; the next day, 2% BSA was added for blocking at room temperature for 1 hour; the experimental group added the amplified phages after each round of panning, and the control group added an equal amount of wild-type phages, incubated at room temperature for 2 hours; washed 10 times with PBST to remove unbound phages; HRP-labeled anti-M13 antibody was added and incubated at room temperature for 1 hour; color development solution was added, and the reaction was protected from light for 60 minutes, and the absorbance was measured. The absorbance gradually increased with the number of panning times, indicating that specific antibodies were enriched. ELISA results are attached. Figure 4 .

[0056] ⑤ Identification of single domain antibody positive clones specific for alpaca IgG2b Fc The experimental group ELISA plate was coated with 100 ng of alpaca IgG2b Fc antigen, and the control group ELISA plate was coated with 100 uL PBS and incubated at 4 ℃ overnight; the phage-coated plates obtained in the third and last rounds of screening were taken, 1056 monoclones were randomly selected in 1 mL of culture medium, cultured at 37 ℃ to the logarithmic phase, and M13 was added for infection at room temperature for 30 min, and then centrifuged to replace fresh culture medium; the next day, the supernatant was collected by centrifugation; at the same time, the ELISA plate was taken, 2% BSA was added and blocked at room temperature for 2 h; supernatant was added to each well of the experimental group and the control group, incubated at room temperature for 2 h; PBST was washed 5 times, M13 antibody was added, and the reaction was continued for 1 h at room temperature; PBST was washed 3-5 times; TMB substrate was added, reacted for 10 min, and the absorbance value was read on the microplate reader; when the absorbance value was greater than 2 compared with the control well, it was determined to be a positive clone; ELISA verification results showed that 582 positive clones were obtained. The screening results of positive clones are attached. Figure 5 .

[0057] ⑥ Sequence analysis of positive clones The 582 positive clones were sequenced, and the sequencing results showed that 30 nucleotide sequences were obtained. The amino acid sequences were analyzed, and all 30 sequences had a typical single-domain antibody structure, which consisted of a framework region (FR1, FR2, FR3 and FR4) and a complementary determining region (CDR1, CDR2 and CDR3). The unique sequence analysis of the positive clones is shown in the attached figure. Figure 6 .

[0058] ⑦ Verification of unique clones specific for alpaca IgG2b Fc The ELISA plate of the experimental group was coated with 100 ng of alpaca IgG2b Fc antigen, and the ELISA plate of the control group was coated with 100 uL PBS and incubated at 4 ℃ overnight; 2 copies of 30 unique monoclonal clones to be verified were selected in 1 mL culture medium, cultured at 37 ℃ to the logarithmic phase, one copy was infected with M13 at room temperature for 30 min and then centrifuged to replace fresh culture medium; the next day, the supernatant was collected by centrifugation; at the same time, the ELISA plate was taken, and 2% BSA was added to block at room temperature for 2 h; supernatant was added to each well of the experimental group and the control group, and incubated at room temperature for 2 h; PBST was washed 5 times, and M13-HRP secondary antibody was added to the plate for 1 h at room temperature; 1 mM IPTG was added to the other copy for induction overnight; the next day, the bacterial pellet was collected by centrifugation, and after breaking, it was centrifuged at 5,000 g for 15 min and the supernatant was collected; at the same time, the ELISA plate was taken, and 2% BSA was added to block at room temperature for 2 h; the monoclonal supernatant was added to each well of the experimental group and the control group, and incubated at room temperature for 2 h; wash 5 times with PBST, add VHH-HRP secondary antibody, incubate at room temperature for 1 h; wash 3-5 times with PBST after the secondary antibody incubation; add TMB substrate, react for 10 min, read the absorbance on the microplate reader; when the absorbance ratio of the control well is greater than 2, it is determined to be a positive clone; ELISA verification results show that the secondary verification results of 30 unique clones are all positive. This shows that all 30 positive sequences can specifically bind to alpaca IgG2b Fc antigen. The verification results of unique clones are attached Figure 7 and attached Figure 8 .

[0059] The present invention successfully screened a single-domain antibody sequence that specifically binds to alpaca IgG2b Fc through phage display technology, and analyzed the binding force of the alpaca IgG2b Fc single-domain antibody through the ELISA method.

[0060] Example 1: Polyclonal phage ELISA to detect the enrichment of alpaca IgG2b Fc-specific antibodies The enrichment of alpaca IgG2bFc antibodies in the original library and the library rescued after the 1st, 2nd, 3rd, and 4th rounds of enrichment was detected by polyclonal phage ELISA. The experimental group was coated with 100 ng of alpaca IgG2b Fc on the ELISA plate, and the blank group was coated with PBS on the ELISA plate without antigen, and incubated at 4 ℃ overnight; the next day, 2% BSA was added to block at room temperature for 1 h; the phage supernatant collected from the library rescued in each round of the alpaca IgG2b Fc selection process and the original library was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 h; PBST was washed 10 times, and the secondary antibody was added at room temperature for 1 h; TMB substrate was added, and the reaction was 10-20 min, and the OD 450 absorbance was read on the microplate reader. The results showed that after 4 rounds of selection, the antibody that specifically binds to alpaca IgG2b Fc was effectively enriched (see Appendix Figure 4).

[0061] Example 2: Analysis of the binding capacity of alpaca IgG2b Fc single domain antibody by ELISA The experimental group used 100 ng of alpaca IgG2b Fc protein to coat the ELISA plate, and the blank group used PBS to coat the ELISA plate, and incubated at 4 ℃ overnight; the next day, 2% BSA was added for blocking at room temperature for 1 hour; the M13 monoclonal supernatant of alpaca IgG2b Fc was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 hours; PBST was washed 10 times, and the secondary antibody was added for 1 hour at room temperature; the substrate was added, reacted for 10-20 minutes, and the absorbance value was read on the ELISA instrument. The ELISA test results showed that the alpaca IgG2b Fc single domain antibody had a good specific binding to the alpaca IgG2b Fc antigen, and the signal value of the experimental group was far greater than that of the blank group (see Appendix Figure 7 ).

[0062] Example 3: Analysis of the binding capacity of alpaca IgG2b Fc single domain antibody by ELISA analysis of periplasmic expression products The experimental group used 100 ng of alpaca IgG2b Fc protein to coat the ELISA plate, and the blank group used PBS to coat the ELISA plate, and incubated at 4 ℃ overnight; the next day, 2% BSA was added for blocking at room temperature for 1 hour; the periplasmic expression product of alpaca IgG2b Fc induced by IPTG was taken as the primary antibody for incubation, and the antibody was added to the control group and the experimental group respectively, and incubated at room temperature for 2 hours; PBST was washed 10 times, and the secondary antibody was added for 1 hour at room temperature; the substrate was added, and the reaction was 10-20 minutes, and the absorbance value was read on the ELISA instrument. The ELISA test results showed that the alpaca IgG2bFc single domain antibody had a good specific binding to the alpaca IgG2b Fc antigen, and the signal value of the experimental group was far greater than that of the blank group (see Appendix Figure 8 ).

[0063] Another aspect of the present application provides a fusion protein comprising the single domain antibody against alpaca IgG2b Fc tag as described above.

[0064] Furthermore, the present application also provides a product, which contains the single-domain antibody with anti-alpaca IgG2b Fc tag as described above or the fusion protein as described above, wherein the product is an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to sites expressing alpaca IgG2b Fc.

[0065] This application uses alpaca IgG2b Fc (alpaca IgG2b Fc) recombinant protein antigen to immunize camels and successfully screens out alpaca IgG2b Fc single-domain antibodies with high affinity activity. The heavy chain variable region of a single-domain antibody has three CDR regions. Among them, CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, single-domain antibodies have higher antigen binding specificity and affinity than traditional antibodies. In addition, in response to the problem that "the existing technology for the development of alpaca IgG2b Fc (alpaca IgG2b Fc) antibodies focuses on monoclonal traditional antibodies, traditional monoclonal antibody screening methods are time-consuming and labor-intensive, traditional antibodies cannot be expressed in prokaryotic systems, have large molecular weights and complex structures, poor tissue permeability, long research and development cycles, high production costs, and large batch-to-batch differences, which seriously limit the development of alpaca IgG2b Fc antibody drugs in my country and cannot meet the diagnosis and treatment needs of patients in my country", this application provides a single-domain antibody based on an anti-alpaca IgG2b Fc tag and its application, which has a short research and development cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost.

[0066] It should be noted that the present invention is not limited to the above-mentioned embodiments. Based on the creative spirit of the present invention, those skilled in the art may also make other changes. These changes made based on the creative spirit of the present invention should be included in the scope of protection required by the present invention.

Claims

1. A single domain antibody against alpaca IgG2b Fc tag, characterized in that: The single domain antibody has the following complementarity determining regions: CDR1, CDR2 and CDR3; Among them, the amino acid sequence of CDR1 is shown in any one of SEQ ID NOs.31-60, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs.61-90, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs.91-120.

2. The single domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that The complementary determining region of the single domain antibody against alpaca IgG2b Fc tag is shown in any one of the following (1)-(30): (1) CDR1 is shown in SEQ ID NO.31, CDR2 is shown in SEQ ID NO.61, and CDR3 is shown in SEQ ID NO.91; (2) CDR1 is shown in SEQ ID NO.32, CDR2 is shown in SEQ ID NO.62, and CDR3 is shown in SEQ ID NO.92; (3) CDR1 is shown in SEQ ID NO.33, CDR2 is shown in SEQ ID NO.63, and CDR3 is shown in SEQ ID NO.93; (4) CDR1 is shown in SEQ ID NO.34, CDR2 is shown in SEQ ID NO.64, and CDR3 is shown in SEQ ID NO.94; (5) CDR1 is shown in SEQ ID NO.35, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO.95; (6) CDR1 is shown in SEQ ID NO.36, CDR2 is shown in SEQ ID NO.66, and CDR3 is shown in SEQ ID NO.96; (7) CDR1 is shown in SEQ ID NO.37, CDR2 is shown in SEQ ID NO.67, and CDR3 is shown in SEQ ID NO.97; (8) CDR1 is shown in SEQ ID NO.38, CDR2 is shown in SEQ ID NO.68, and CDR3 is shown in SEQ ID NO.98; (9) CDR1 is shown in SEQ ID NO.39, CDR2 is shown in SEQ ID NO.69, and CDR3 is shown in SEQ ID NO.99; (10) CDR1 is shown in SEQ ID NO.40, CDR2 is shown in SEQ ID NO.70, and CDR3 is shown in SEQ ID NO.100; (11) CDR1 is shown in SEQ ID NO.41, CDR2 is shown in SEQ ID NO.71, and CDR3 is shown in SEQ ID NO.101; (12) CDR1 is shown in SEQ ID NO.42, CDR2 is shown in SEQ ID NO.72, and CDR3 is shown in SEQ ID NO.102; (13) CDR1 is shown in SEQ ID NO.43, CDR2 is shown in SEQ ID NO.73, and CDR3 is shown in SEQ ID NO.103; (14) CDR1 is shown in SEQ ID NO.44, CDR2 is shown in SEQ ID NO.74, and CDR3 is shown in SEQ ID NO.104; (15) CDR1 is shown in SEQ ID NO.45, CDR2 is shown in SEQ ID NO.75, and CDR3 is shown in SEQ ID NO.105; (16) CDR1 is shown in SEQ ID NO.46, CDR2 is shown in SEQ ID NO.76, and CDR3 is shown in SEQ ID NO.106; (17) CDR1 is shown in SEQ ID NO.47, CDR2 is shown in SEQ ID NO.77, and CDR3 is shown in SEQ ID NO.107; (18) CDR1 is shown in SEQ ID NO.48, CDR2 is shown in SEQ ID NO.78, and CDR3 is shown in SEQ ID NO.108; (19) CDR1 is shown in SEQ ID NO.49, CDR2 is shown in SEQ ID NO.79, and CDR3 is shown in SEQ ID NO.109; (20) CDR1 is shown in SEQ ID NO.50, CDR2 is shown in SEQ ID NO.80, and CDR3 is shown in SEQ ID NO.110; (21) CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.81, and CDR3 is shown in SEQ ID NO.111; (22) CDR1 is shown in SEQ ID NO.52, CDR2 is shown in SEQ ID NO.82, and CDR3 is shown in SEQ ID NO.112; (23) CDR1 is shown in SEQ ID NO.53, CDR2 is shown in SEQ ID NO.83, and CDR3 is shown in SEQ ID NO.113; (24) CDR1 is shown in SEQ ID NO.54, CDR2 is shown in SEQ ID NO.84, and CDR3 is shown in SEQ ID NO.114; (25) CDR1 is shown in SEQ ID NO.55, CDR2 is shown in SEQ ID NO.85, and CDR3 is shown in SEQ ID NO.115; (26) CDR1 is shown in SEQ ID NO.56, CDR2 is shown in SEQ ID NO.86, and CDR3 is shown in SEQ ID NO.116; (27) CDR1 is shown in SEQ ID NO.57, CDR2 is shown in SEQ ID NO.87, and CDR3 is shown in SEQ ID NO.117; (28) CDR1 is shown in SEQ ID NO.58, CDR2 is shown in SEQ ID NO.88, and CDR3 is shown in SEQ ID NO.118; (29) CDR1 is shown in SEQ ID NO.59, CDR2 is shown in SEQ ID NO.89, and CDR3 is shown in SEQ ID NO.119; (30) CDR1 is shown in SEQ ID NO.60, CDR2 is shown in SEQ ID NO.90, and CDR3 is shown in SEQ ID NO.

120.

3. The single domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that: The single domain antibody against alpaca IgG2b Fc tag has the following framework regions: FR1, FR2, FR3, FR4; in, The amino acid sequence of FR1 is shown in any one of SEQ ID NOs. 121-150; The amino acid sequence of FR2 is shown in any one of SEQ ID NOs. 151-180; The amino acid sequence of FR3 is shown in any one of SEQ ID NOs. 181-210; The amino acid sequence of FR4 is shown in any one of SEQ ID NOs. 211-240.

4. The single domain antibody against alpaca IgG2b Fc tag according to claim 3, characterized in that: The framework region of the single domain antibody is as shown in any one of the following (31)-(60): (31) FR1 is shown in SEQ ID NO.121, FR2 is shown in SEQ ID NO.151, FR3 is shown in SEQ ID NO.181; FR4 is shown in SEQ ID NO.211; (32) FR1 is shown in SEQ ID NO.122, FR2 is shown in SEQ ID NO.152, FR3 is shown in SEQ ID NO.182; FR4 is shown in SEQ ID NO.212; (33) FR1 is shown in SEQ ID NO.123, FR2 is shown in SEQ ID NO.153, FR3 is shown in SEQ ID NO.183; FR4 is shown in SEQ ID NO.213; (34) FR1 is shown in SEQ ID NO.124, FR2 is shown in SEQ ID NO.154, FR3 is shown in SEQ ID NO.184; FR4 is shown in SEQ ID NO.214; (35) FR1 is shown in SEQ ID NO.125, FR2 is shown in SEQ ID NO.155, FR3 is shown in SEQ ID NO.185; FR4 is shown in SEQ ID NO.215; (36) FR1 is shown in SEQ ID NO.126, FR2 is shown in SEQ ID NO.156, FR3 is shown in SEQ ID NO.186; FR4 is shown in SEQ ID NO.216; (37) FR1 is shown in SEQ ID NO.127, FR2 is shown in SEQ ID NO.157, FR3 is shown in SEQ ID NO.187; FR4 is shown in SEQ ID NO.217; (38) FR1 is shown in SEQ ID NO.128, FR2 is shown in SEQ ID NO.158, FR3 is shown in SEQ ID NO.188; FR4 is shown in SEQ ID NO.218; (39) FR1 is shown in SEQ ID NO.129, FR2 is shown in SEQ ID NO.159, FR3 is shown in SEQ ID NO.189; FR4 is shown in SEQ ID NO.219; (40) FR1 is shown in SEQ ID NO.130, FR2 is shown in SEQ ID NO.160, FR3 is shown in SEQ ID NO.190; FR4 is shown in SEQ ID NO.220; (41) FR1 is shown in SEQ ID NO.131, FR2 is shown in SEQ ID NO.161, FR3 is shown in SEQ ID NO.191; FR4 is shown in SEQ ID NO.221; (42) FR1 is shown in SEQ ID NO.132, FR2 is shown in SEQ ID NO.162, FR3 is shown in SEQ ID NO.192; FR4 is shown in SEQ ID NO.222; (43) FR1 is shown in SEQ ID NO.133, FR2 is shown in SEQ ID NO.163, FR3 is shown in SEQ ID NO.193; FR4 is shown in SEQ ID NO.223; (44) FR1 is shown in SEQ ID NO.134, FR2 is shown in SEQ ID NO.164, FR3 is shown in SEQ ID NO.194; FR4 is shown in SEQ ID NO.224; (45) FR1 is shown in SEQ ID NO.135, FR2 is shown in SEQ ID NO.165, FR3 is shown in SEQ ID NO.195; FR4 is shown in SEQ ID NO.225; (46) FR1 is shown in SEQ ID NO.136, FR2 is shown in SEQ ID NO.166, FR3 is shown in SEQ ID NO.196; FR4 is shown in SEQ ID NO.226; (47) FR1 is shown in SEQ ID NO.137, FR2 is shown in SEQ ID NO.167, FR3 is shown in SEQ ID NO.197; FR4 is shown in SEQ ID NO.227; (48) FR1 is shown in SEQ ID NO.138, FR2 is shown in SEQ ID NO.168, FR3 is shown in SEQ ID NO.198; FR4 is shown in SEQ ID NO.228; (49) FR1 is shown in SEQ ID NO.139, FR2 is shown in SEQ ID NO.169, FR3 is shown in SEQ ID NO.199; FR4 is shown in SEQ ID NO.229; (50) FR1 is shown in SEQ ID NO.140, FR2 is shown in SEQ ID NO.170, FR3 is shown in SEQ ID NO.200; FR4 is shown in SEQ ID NO.230; (51) FR1 is shown in SEQ ID NO.141, FR2 is shown in SEQ ID NO.171, FR3 is shown in SEQ ID NO.201; FR4 is shown in SEQ ID NO.231; (52) FR1 is shown in SEQ ID NO.142, FR2 is shown in SEQ ID NO.172, FR3 is shown in SEQ ID NO.202; FR4 is shown in SEQ ID NO.232; (53) FR1 is shown in SEQ ID NO.143, FR2 is shown in SEQ ID NO.173, FR3 is shown in SEQ ID NO.203; FR4 is shown in SEQ ID NO.233; (54) FR1 is shown in SEQ ID NO.144, FR2 is shown in SEQ ID NO.174, FR3 is shown in SEQ ID NO.204; FR4 is shown in SEQ ID NO.234; (55) FR1 is shown in SEQ ID NO.145, FR2 is shown in SEQ ID NO.175, FR3 is shown in SEQ ID NO.205; FR4 is shown in SEQ ID NO.235; (56) FR1 is shown in SEQ ID NO.146, FR2 is shown in SEQ ID NO.176, FR3 is shown in SEQ ID NO.206; FR4 is shown in SEQ ID NO.236; (57) FR1 is shown in SEQ ID NO.147, FR2 is shown in SEQ ID NO.177, FR3 is shown in SEQ ID NO.207; FR4 is shown in SEQ ID NO.237; (58) FR1 is shown in SEQ ID NO.148, FR2 is shown in SEQ ID NO.178, FR3 is shown in SEQ ID NO.208; FR4 is shown in SEQ ID NO.238; (59) FR1 is shown in SEQ ID NO.149, FR2 is shown in SEQ ID NO.179, FR3 is shown in SEQ ID NO.209; FR4 is shown in SEQ ID NO.239; (60) FR1 is shown in SEQ ID NO.150, FR2 is shown in SEQ ID NO.180, FR3 is shown in SEQ ID NO.210; FR4 is shown in SEQ ID NO.

240.

5. The single domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that: The amino acid sequence of the single domain antibody is shown in any one of SEQ ID NOs. 1-30.

6. The single domain antibody against alpaca IgG2b Fc tag according to claim 4, characterized in that The framework region is a heavy chain framework region, and at least a portion of the heavy chain framework region is independently derived from an alpaca-derived antibody.

7. The single domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that: The single-domain antibody against alpaca IgG2b Fc tag is prepared by immunizing a camel with an alpaca IgG2b Fc recombinant protein antigen, collecting peripheral blood cells of the immunized camel, separating alpaca IgG2b Fc affinity lymphocytes therefrom, extracting total RNA and reversely transcribing it into cDNA, cloning the V region of the camel heavy chain antibody by using the Nest-PCR technology, inserting the V region into the phage plasmid pMES4, constructing a phage expression library, and then performing multiple rounds of screening on the alpaca IgG2b Fc antigen by using the phage display technology, and verifying the binding force of the obtained single-domain antibody by an enzyme-linked immunosorbent assay, thereby screening out a single-domain antibody against alpaca IgG2bFc tag with high affinity activity.

8. The single domain antibody against alpaca IgG2b Fc tag according to claim 1, characterized in that: The DNA sequence of the single domain antibody against alpaca IgG2b Fc tag is shown in any one of SEQ ID NOs. 241-270.

9. A fusion protein, characterized in that: It contains the single domain antibody against alpaca IgG2b Fc tag as described in any one of claims 1-8.

10. A product, characterized in that It contains the single-domain antibody with an anti-alpaca IgG2b Fc tag as described in any one of claims 1 to 8 or the fusion protein as described in claim 9, and the product is an in vitro diagnostic reagent, an anti-tumor drug, an immune cell for cell immunotherapy, or a carrier for delivering drugs to a site expressing alpaca IgG2b Fc.

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